Adenylate cyclase systems consist of inhibitory and stimulatory receptors linked to a catalytic unit through inhibitory and stimulatory proteins, Gi and Gs, respectively. Similarly, the retinal photon receptor rhodopsin is linked with a phosphodiesterase through transducin (T), which is similar to Gs and Gi. Each has a guanyl nucleotide-binding Alpha subunit (45 kDa Gs, 41 kDa Gi, and 39 kDa T) and Beta, Gamma subunit (35 and 10kDa). (1) The Alpha and BetaGamma subunits of Gi, Gs, and T were purified. (2) To demonstrate the functional similarity between Gi and T, subunits of Gi were tested for their ability to replace the corresponding T subunits in the rhodopsin-stimulated GTPase reaction. GTPase activity was observed when the Alpha subunit of Gi or T was combined with BetaGamma subunits of either Gi or T and rhodopsin. These data demonstrate (a) that GiAlpha possesses intrinsic GTPase activity and (b) that photolyzed rhodopsin can mimic an inhibitory receptor-agonist complex in its ability to interact with Gi. (3) The purified Alpha subunits of Gi and T are substrates for the pertussis toxin-catalyzed ADP-ribosylation reaction. ADP-ribosylation of GiAlpha by pertussis toxin was much more effective in the presence of GBetaGamma. Photolyzed rhodopsin mimicking agonist-inhibitory receptor complex inhibited the reaction. ADP-ribosylation of GiAlpha with GBetaGamma and photolyzed rhodopsin was enhanced by GDP and GDPBetaS but not by Gpp(NH)p, Gpp(CH-2)p, or GTPGammaS; dark rhodopsin had only minimal effects on ADP-ribosylation. Since dark rhodopsin and guanosine diphosphate analogues favor the associated inactive GiAlpha-GBetaGamma species, whereas photolyzed rhodopsin and nucleoside triphosphate analogues promote dissociation of GiAlpha GBetaGamma to release the active forms, GiAlpha and GBetaGamma, these studies support the hypothesis that the preferred substrate for pertussis toxin is the inactive species, GiAlpha GBetaGamma.